Viral load

Viral load, also known as viral burden, viral titre or viral titer, is a numerical expression of the quantity of virus in a given volume. It is often expressed as viral particles, or infectious particles per mL depending on the type of assay. A higher viral burden, titre, or viral load often correlates with the severity of an active viral infection. The quantity of virus / mL can be calculated by estimating the live amount of virus in an involved body fluid. For example, it can be given in RNA copies per millilitre of blood plasma. Tracking viral load is used to monitor therapy during chronic viral infections, and in immunocompromised patients such as those recovering from bone marrow or solid organ transplantation. Currently, routine testing is available for HIV-1, cytomegalovirus, hepatitis B virus, and hepatitis C virus.

Technologies for viral load testing

A recent review study by Puren et al.[1] categorizes viral load testing into three types: (1) nucleic acid amplification based tests (NATs or NAATs) commercially available in the United States with Food and Drug Administration (FDA) approval, or on the market in the European Economic Area (EEA) with the CE marking; (2) "Home–brew" or in-house NATs; (3) non-nucleic acid-based test.

Nucleic acid-based tests (NATs)

There are many different molecular based test methods for quantifying the viral load using NATs.[2] The starting material for amplification can be used to divide these molecular methods into three groups:

  1. Target amplification which uses the nucleic acid itself. Just a few of the more common methods
    • The Polymerase Chain Reaction ((PCR) method of in vitro DNA synthesis uses a DNA template, polymerase, buffers, primers, and nucleotides to multiply the HIV in the blood sample. Then a chemical reaction marks the virus. The markers are measured and used to calculate the amount of virus. PCR is used to quantify integrated DNA
    • Reverse Transcription Polymerase Chain Reaction (RT-PCR) is a variation of PCR that can be used to quantify viral RNA. RNA is used as the starting material for this method and converted to double-stranded DNA, using the enzyme reverse transcriptase (RT)
    • The Nucleic Acid Sequence Based Amplification (NASBA) method is a transcription-based amplification system (TAS) variation of PCR. RNA is used as the target and a DNA copy is made. The DNA copy is then transcribed into RNA and amplified. Several TAS commercial variations are available including; transcription-mediated amplification (TMA), and self-sustaining sequence replication (3SR)
  2. Probe specific amplification uses synthetic probes that preferentially bind to a target sequence. The probes are then amplified
  3. Signal amplification uses large amounts of signal bound to an unamplified target originally present in the sample. One commonly used method:
    • The branched DNA (bDNA) method can use either DNA or RNA as the target nucleic acid. Short probes attached to a solid support and capture the target nucleic acid. Additional extender probes also bind to the target nucleic acid and to numerous reporter molecules which are used to increase the signal intensity, which is converted to a viral count.
HIV NATs for viral load approved & licensed by the FDA for sale in the United States[3]
Test name Molecular method Use Approved Manufacturer
Roche Amplicor HIV-1 Monitor Test[4] PCR Viral load 3/2/1999 Roche Molecular Systems, Inc. Pleasanton, CA
NucliSens HIV-1 QT[5] NASBA Viral load 11/19/2001 bioMerieux, Inc Durham, NC
Trugene HIV-1 Genotyping Kit and Open Gene DNA Sequencing System[6] HIV-1 Genotyping Patient monitoring 4/24/2002 Siemens Medical Solutions Diagnostics Berkeley, CA
ViroSeq HIV-1 Genotyping System with the 3700 Genetic Analyzer[7] HIV-1 Genotyping Patient monitoring 6/11/2003 Celera Diagnostics Alameda, CA
Versant HIV-1 RNA 3.0[8] bDNA Patient monitoring 9/11/2002 Siemens Medical Solutions Diagnostics Berkeley, CA
Procleix Ultrio Assay[9] TMA (commercial variation of NASBA)

The three steps to the Procleix Ultrio Assay are done in one tube.[10] The first step is specimen preparation, the second is transcription-mediated amplification (TMA), and the third is a hybridization protection assay (HPA) using single-stranded complementary chemiluminescent labeled probes. A luminometer is used to measure the signal. The sensitivity of this assay is 98%.

 Qualitative detection of HIV-1 RNA and hepatitis C virus (HCV) RNA from volunteer donors of whole blood and blood components, screen of live organ donors, and test blood specimens to screen cadaveric donors. HBV screening of individual samples and pooled samples. 10/3/2006 Gen-Probe San Diego, CA US Licence 1592 Chiron Corporation
Human Immunodeficiency Virus, Type 1 (HIV-1) Reverse Transcription (RT) Polymerase Chain Reaction (PCR) Assay[11] PCR Qualitative detection of HIV-1 RNA in pools of human Source Plasma 1/31/2007 BioLife Plasma Services, L.P. Deerfield, IL US Licence 1640
Abbott RealTime HIV-1 Amplification Kit[12] RT-PCR

The sensitivity (linear range) for this assay is 40 copies/mL to 1010 copies/mL. Two probes are used. The HIV-1 probe is labeled with a fluorescent molecule and covalently binds to the 5’ end. The second probe is a short oligonucleotide with a 3’ end quencher molecule attached complementary to the 5’ end of the HIV-1 probe. If the HIV-1 probe finds and attaches to a HIV target the quencher molecule is released and the resulting fluorescent emission is measured. The fluorescence is proportional to the log of the amount of virus in the sample.

Quantitation of HIV-1 5/11/2007 ABBOTT Molecular, Inc Des Plaines, IL
COBAS AmpliPrep/COBAS TaqMan HIV-1 Test[13] PCR

Uses fluorescent resonance energy transfer (FRET) to enhance its automated RT-PCR. In the FRET reaction, a donor and acceptor probe exchange excitation energy when within 1-5 base pairs (bp) on the target sequences. The energy is emitted in the form of heat or fluorescence. The probes are designed to bind 1-5 bp from each other. The energy emission is proportional to the concentration of viral particles. The linear range for this assay is 48 copies/mL of blood.

 Quantitation of HIV-1 5/11/2007 Roche Molecular Systems, Inc Pleasanton, CA

Non-nucleic acid-based tests

ExaVir™ Load Version 3 from Cavidi AB is a largely manual test, which has the European regulatory approval (CE-Mark) for clinical use and is also used for viral load monitoring. Virus-associated reverse transcriptase (RT) activity is measured and can therefore detect all types and subtypes of HIV.[14] The technology does not require sophisticated laboratories.[15][16]

Components for viral load tests

Specimens

EDTA plasma is the best source of cell-free viral RNA for RNA-based viral load testing. Consideration of specimen collection, storage and biosafety measures is essential. Extraction of RNA from plasma requires specialized equipment, reagents and training, placing it out of reach for medium to small labs with limited resources. A large sample (> 1 mL of plasma) is needed for a linear range bottoming out at 50 copies/mL, requiring venipuncture. This linear range is best for treatment monitoring. If a higher linear range of more than 1000 copies/mL is acceptable, a finger stick would supply a sufficient specimen for diagnosis of HIV infection during infancy.

EDTA plasma can be stored at room temperature for 30 hours, 14 days at 4 °C and extended periods of time at -70 °C without significant decreases in viral load signal. The RNA in smaller blood specimens, such as dried plasma spots (DPS) or dried blood spots (DBS) from finger sticks is reportedly stable at room temperature periods ranging from 4 weeks to 1 year. The virus is inactivated in dried samples, reducing the danger from specimen handling. DBS and DPS were successfully evaluated for viral load testing, but their linear range is 3 log10 or 4 log10 copies/mL. Because of this lack of sensitivity, dried specimens are useful for HIV screening but not for viral load determination.

Measuring

Viral load is typically reported as copies of HIV in a milliliter (mL) of blood. Changes in viral load are usually reported as a log change (in powers of 10). For example, a three log increase in viral load (3 Log10) is an increase of 103 or 1000 times the previously reported level, while a drop from 500,000 to 500 copies would be a three-log-drop (also 3 Log10).

Diagnosis

The window period for a test is the amount of time from the initial infection event until the disease can be detected.[17] Exposure to HIV, followed by replication of the virus, may take as long as six months to reach a level detectable in many testing methods. An HIV antibody test usually detects the HIV antibodies within two to eight weeks, but can have a valid negative result for a long as 2 to 6 months after initial infection. Viral load tests can also be used to diagnose HIV infection, especially in children under 18 months born to mothers with HIV, where the presence of maternal antibodies prevents the use of antibody-based (ELISA) diagnostic tests.[18] Pooled viral RNA testing shortens the window period to a median of 17 days (95% CI, 13-28 Days).[19] Although it is not the standard of care to use this test for diagnosis, in communities with high HIV prevalence, this test has a significantly improved negative predictive value over 3rd and 4th generation tests for detecting acute HIV infections.[20][21]

On June 15, 2010, the FDA approved the first diagnostic test capable of detecting HIV antigens and HIV antibodies. The Abbott ARCHITECT HIV Ag/Ab combo test, a fourth-generation test, has an increased sensitivity for detecting infections during the acute phase (when compared to 1st and 3rd generation tests), when the immune system is still developing antibodies and the virus is replicating unchecked, and in one study, was able to detect 83% of such infections.[22] A person, who may be unaware of the infection, is highly infectious during this time yet may test negative for HIV using tests that detect anti-HIV antibodies only. Although Nucleic Acid Amplification Testing NAAT is more expensive and can take a week for processing, some have argued that it may still be a preferred way to screen for HIV.[23]

Prognosis

Viral load is used to predict how long an individual will remain healthy, or how quickly the disease will progress. A viral load greater than 100,000 copies/mL of blood within six months of seroconversion indicates a greater likelihood of developing AIDS within five years. A viral load less than 10,000 copies/mL of blood in the early stages indicates a decreased risk of developing AIDS.

Therapy

Treatment guidelines recommend that anyone with a viral load greater than 100,000 copies/mL of blood should begin treatment. HIV is a retrovirus, an RNA virus that enters a host cell and uses the host DNA replication machinery and the enzyme reverse transcriptase to produce DNA from the viral RNA genome. HIV also produces an integrase enzyme which is used to integrate the newly produced viral DNA into the host’s DNA. The virus is then replicated every time the host cell's DNA replicates. Due to the nature of the virus the drugs used to treat HIV are called antiretroviral medicines, and the course of treatment is called antiretroviral therapy (ART). These potent medicines cannot cure an individual; they can however manage the virus and slow the progression of the HIV infection. Strict compliance with the prescribed ART regimen is vital to controlling the disease.

Highly active antiretroviral therapy (HAART) is the current recommended treatment for HIV. HAART entails taking a combination (regimen) of three or more ART medications from at least two different classes of drugs. There are six classes of ART medications:

Each class of medications uses a different mode of action to blocks the virus. Treatment is more effective in controlling the virus when a combination of medications from different classes is used. HAART also reduces the risk of developing drug resistance. Viral load tests are used to monitor the effects ART, to track viral suppression, and detect treatment failure. Successful combination ART should give a fall in viral load of 1.5 to 2 logs (30-100 fold) within six weeks, with the viral load falling below the limit of detection within four to six months.[24]

Laboratory monitoring schedule for patients using ART:

Initial Physician visit Prior to beginning ART Beginning or modifying ART 2 – 8 weeks after beginning or modifying ART Every 3 – 6 months Every 6 – 12 months Other
Viral load When entering into care Every 3 – 6 months Baseline for comparison Repeat every 4 – 8 weeks until viral load is suppressed to <200 copies/mL blood, then every 3 – 6 months Individuals with a suppressed viral load, who are clinically & immunologically stable for more than 2 – 3 years, may go to every 6 months Treatment failure or when clinically indicated
CD4 When entering into care Every 3 – 6 months Baseline for comparison Routine monitoring Individuals with a suppressed viral load, who are clinically stable, CD4 count monitoring may go to every 6 – 12 months Treatment failure or when clinically incicated

Blips

While receiving ART some patients with undetectable viral load measurements may experience an increase in viral load, to a low level (usually below 400 copies/mL blood), and then returned to an undetectable level. These transient blips do not indicate that the virus is developing resistance to drug therapy. Blips appear to be more common in the winter, suggesting a connection with illness such as colds and influenza.[25] Viral load blips are partially explained by various patient related factors, and thought to be relatively common. High level and sustained increases in viral load are frequently related to the development of drug resistance and/or viral mutations, and often dictate changes in ART.

Contagiousness

Viral load is a good predictor of the likelihood of transmitting an active virus between the same or different species. When a virus crosses between species it is called zoonosis.

The higher the viral load value, the more viral elements there are in blood, other body fluids or carcass. For example, individuals with HIV are most contagious during the earliest (acute) stages of the infection, sometimes with millions of copies of HIV per centiliter of blood. According to one estimate, the majority of transmissions among gay men in the UK occur during primary infection.[26] This is because, at this phase, the immune response is still developing. Antibody levels against the virus during acute infection are often too low to be detected, meaning that an antibody test for a highly infectious individual can come back negative.

There is at least one documented case of an HIV-positive individual with an undetectable viral load infecting his partner.[27] Nevertheless, two recent major studies of serodiscordant couples in which the positive partner had an undetectable viral load have found no linked transmission events despite at least some unprotected sex among the couples.[28][29] HPTN 052, a third study, found one linked transmission event in a test group of 886 couples,[30] although it is unclear whether the originally infected partner in this case had achieved an undetectable viral load at the time of transmission.[31]

Viral load in blood corresponds imperfectly to viral load in other parts of the body. Individuals with undetectable viral loads in their blood might not have undetectable viral loads in other bodily fluids, such as semen or vaginal secretions. In one recent study, of 83 men with undetectable viral loads, 21 had very slight but not undetectable viral loads in their semen.[32] However, it is unclear how much this actually increases transmission risk.

Other factors that affect viral load

Different test methods often give different results for the same patient sample. To be comparable the same test method (Target amplification, probe specific amplification, or signal amplification) should be used each time a patient specimen is run. Ideally patient testing should be conducted at the same medical laboratory, using the same viral load test and analyzer. Time of day, fatigue, and stress can also affect viral load values. Recent immunizations or infections can affect the viral load test. Testing should be postponed for at least four weeks after an immunization or infection.

CD4 white blood cells

CD4 cells are the primary target of HIV. A CD4 test quantifies Helper T cells and is often combined with viral load testing to monitor the progression of HIV. CD4 testing shows the strength of the immune system, but does not report viral activity. As established by the Centers for Disease Control and Prevention (CDC), a person with HIV and a CD4 count below 200 or a CD4 percentage below 14% is considered to have AIDS.[33] An increased CD4 count can result from an immune response to an infection or a recent vaccination. A decreased CD4 count, in combination with higher numbers on a viral load test, indicates an increased risk of getting sick from opportunistic diseases.

References

  1. ↑ Puren, Adrian; Gerlach, Jay L.; Weigl, Bernhard H.; Kelso, David M.; Domingo, Gonzalo J. (2010). "Laboratory Operations, Specimen Processing, and Handling for Viral Load Testing and Surveillance". The Journal of Infectious Diseases 201: S27–36. doi:10.1086/650390. PMID 20225943.
  2. ↑ Buckingham L, Flaws ML. 2007. Molecular Diagnostics Fundamentals, Methods, & Clinical Applications. F. A Davis Company. Philadelphia
  3. ↑ Complete List of Donor Screening Assays for Infectious Agents and HIV Diagnostic Assays. US Food & Drug Administration. November 30, 2010. (available to download from http://www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/UCM080466
  4. ↑ Consumer Affairs Branch (CBER) (2010-08-17). "Roche Amplicor HIV-1 Monitor Test". Fda.gov. Retrieved 2012-10-16.
  5. ↑ Consumer Affairs Branch (CBER). "NucliSens HIV-1 QT". Fda.gov. Retrieved 2012-10-16.
  6. ↑ "TRUGENE® HIV-1 Genotyping Kit and OpenGene® DNA Sequencing System- BK110007". Fda.gov. 2011-03-10. Retrieved 2012-10-16.
  7. ↑ "ViroSeq HIV-1 Genotyping System with the 3700 Genetic Analyzer". Fda.gov. Retrieved 2012-10-16.
  8. ↑ Consumer Affairs Branch (CBER). "VERSANT HIV-1 RNA 3.0 Assay (bDNA)". Fda.gov. Retrieved 2012-10-16.
  9. ↑ Consumer Affairs Branch (CBER). "Procleix Ultrio Plus Assay". Fda.gov. Retrieved 2012-10-16.
  10. ↑ Josko, D (2010). "Molecular virology in the clinical laboratory". Clinical laboratory science 23 (4): 231–6. PMID 21140798.
  11. ↑ "Human Immunodeficiency Virus, Type 1 (HIV-1) Reverse Transcription (RT) Polymerase Chain Reaction (PCR) Assay". Fda.gov. 2007-01-31. Retrieved 2012-10-16.
  12. ↑ Consumer Affairs Branch (CBER) (2009-02-18). "Abbott RealTime HIV-1 Amplification Reagent Kit, Abbott RealTime HIV-1 Calibrator Kit, Abbott RealTime HIV-1 Control Kit". Fda.gov. Retrieved 2012-10-16.
  13. ↑ Consumer Affairs Branch (CBER) (2009-02-19). "COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, 48 Tests; COBAS AmpliPrep/COBAS TaqMan Wash Reagent, 5.1 L". Fda.gov. Retrieved 2012-10-16.
  14. ↑ Malmsten, Anders; Shao, Xing-Wu; Sjödahl, Staffan; Fredriksson, Eva-Lena; Pettersson, Ingvar; Leitner, Thomas; Källander, Clas F.R.; Sandström, Eric; Gronowitz, J. Simon (2005). "Improved HIV-1 viral load determination based on reverse transcriptase activity recovered from human plasma". Journal of Medical Virology 76 (3): 291–6. doi:10.1002/jmv.20360. PMID 15902697.
  15. ↑ Greengrass, Vicki L.; Turnbull, Shannon P.; Hocking, Jane; Dunne, Amanda L.; Tachedjian, Gilda; Corrigan, Gary E.; Crowe, Suzanne M. (2005). "Evaluation of a Low Cost Reverse Transcriptase Assay for Plasma HIV-1 Viral Load Monitoring". Current HIV Research 3 (2): 183–90. doi:10.2174/1570162053506955. PMID 15853722.
  16. ↑ Jennings, C.; Fiscus, S. A.; Crowe, S. M.; Danilovic, A. D.; Morack, R. J.; Scianna, S.; Cachafeiro, A.; Brambilla, D. J.; et al. (2005). "Comparison of Two Human Immunodeficiency Virus (HIV) RNA Surrogate Assays to the Standard HIV RNA Assay". Journal of Clinical Microbiology 43 (12): 5950–6. doi:10.1128/JCM.43.12.5950-5956.2005. PMC 1317157. PMID 16333081.
  17. ↑ Testing Window Period. (2010). aids.gov. Available at http://aids.gov/hiv-aids-basics/prevention/your-hiv-status/testing-window-period/
  18. ↑ IV. The Challenge of Diagnosis. The United States President’s Emergency Plan for Aids Relief. Available at http://www.pepfar.gov/progress/76907.htm
  19. ↑ Fiebig, E. W.; Wright, D. J.; Rawal, B. D.; Garrett, P. E.; Schumacher, R. T.; Peddada, L; Heldebrant, C; Smith, R; Conrad, A; Kleinman, S. H.; Busch, M. P. (2003). "Dynamics of HIV viremia and antibody seroconversion in plasma donors: Implications for diagnosis and staging of primary HIV infection". AIDS (London, England) 17 (13): 1871–9. doi:10.1097/01.aids.0000076308.76477.b8 (inactive 2015-02-01). PMID 12960819.
  20. ↑ Patel, P.; MacKellar, D.; Simmons, P.; Uniyal, A.; Gallagher, K.; Bennett, B.; Sullivan, T. J.; Kowalski, A.; et al. (2010). "Detecting Acute Human Immunodeficiency Virus Infection Using 3 Different Screening Immunoassays and Nucleic Acid Amplification Testing for Human Immunodeficiency Virus RNA, 2006-2008". Archives of Internal Medicine 170 (1): 66–74. doi:10.1001/archinternmed.2009.445. PMID 20065201.
  21. ↑ Cohen, Myron S.; Gay, Cynthia L.; Busch, Michael P.; Hecht, Frederick M. (2010). "The Detection of Acute HIV Infection". The Journal of Infectious Diseases 202 (S2): S270–7. doi:10.1086/655651. PMID 20846033.
  22. ↑ Chavez P, Wesolowski L, Patel P, Delaney K, Owen SM; Wesolowski; Patel; Delaney; Owen (December 2011). "Evaluation of the performance of the Abbott ARCHITECT HIV Ag/Ab Combo Assay". J. Clin. Virol. 52 Suppl 1: S51–5. doi:10.1016/j.jcv.2011.09.010. PMID 21983253.
  23. ↑ Karris MY, Anderson CM, Morris SR, Smith DM, Little SJ; Anderson; Morris; Smith; Little (June 2012). "Cost savings associated with testing of antibodies, antigens, and nucleic acids for diagnosis of acute HIV infection". J. Clin. Microbiol. 50 (6): 1874–8. doi:10.1128/JCM.00106-12. PMC 3372146. PMID 22442319.
  24. ↑ DHHS Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents. May 4, 2006. (available for download from AIDSInfo)
  25. ↑ Carter M. (2008). Most viral load blips are short-lasting and of no significance. NAM Publications. Available at http://www.aidsmap.com/Most-viral-load-blips-are-short-lasting-and-of-no-significance/page/1430421/
  26. ↑ "HIV & AIDS Information :: Viral load and the risk of transmission - Primary infection and sexual transmission". Aidsmap.com. doi:10.1371/journal.pone.0055312. Retrieved 2015-02-01.
  27. ↑ Stürmer, M; Doerr, H. W.; Berger, A; Gute, P (2008). "Is transmission of HIV-1 in non-viraemic serodiscordant couples possible?". Antiviral Therapy 13 (5): 729–32. PMID 18771057.
  28. ↑ Category: Treatment as Prevention. "CROI 2014: HIV Transmission through Condomless Sex [VIDEO] | Treatment as Prevention". hivandhepatitis.com. Retrieved 2015-02-01.
  29. ↑ Derek Thaczuk Published: 15 November 2006 (2006-11-15). "HIV & AIDS Information :: Spanish find that serodiscordant couples having unprotected sex to conceive - no cases of HIV transmission". Aidsmap.com. Retrieved 2015-02-01.
  30. ↑ Cohen, Myron S.; Chen, Ying Q.; McCauley, Marybeth; Gamble, Theresa; Hosseinipour, Mina C.; Kumarasamy, Nagalingeswaran; Hakim, James G.; Kumwenda, Johnstone; Grinsztejn, Beatriz; Pilotto, Jose H.S.; Godbole, Sheela V.; Mehendale, Sanjay; Chariyalertsak, Suwat; Santos, Breno R.; Mayer, Kenneth H.; Hoffman, Irving F.; Eshleman, Susan H.; Piwowar-Manning, Estelle; Wang, Lei; Makhema, Joseph; Mills, Lisa A.; De Bruyn, Guy; Sanne, Ian; Eron, Joseph; Gallant, Joel; Havlir, Diane; Swindells, Susan; Ribaudo, Heather; Elharrar, Vanessa; et al. (2011). "Prevention of HIV-1 Infection with Early Antiretroviral Therapy". New England Journal of Medicine 365 (6): 493–505. doi:10.1056/nejmoa1105243. PMC 3200068. PMID 21767103.
  31. ↑ "Focusing specifically on the one HIV transmission that occurred in the immediate treatment group, Cohen noted that the HIV-negative partner tested positive for the virus—using standard Western blot testing—on day 85 of the study, whereas the HIV-positive partner’s first undetectable viral load was documented 28 days after entering the study. The researchers believe that the HIV-negative partner was infected at least 50 days before testing positive, likely 84 to 190 days earlier. In other words, the HIV-negative partner was likely infected with the virus before the HIV-positive partner was able to achieve an undetectable viral load while on treatment."
  32. ↑ "Top Stories : Undetectable Viral Load? Not Necessarily in Semen - by Tim Horn". Aidsmeds.com. Retrieved 2015-02-01.
  33. ↑ U.S. Department of Health and Human Services. 2009. HIV and Its Treatment: What You Should Know. Available for download from http://aidsinfo.nih.gov/contentfiles/HIVandItsTreatment_cbrochure_en.pdf
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